New Glycosylated Dihydrochalcones Obtained by Biotransformation of 2′-Hydroxy-2-methylchalcone in Cultures of Entomopathogenic Filamentous Fungi

Flavonoids, including chalcones, are more stable and bioavailable in the form of glycosylated and methylated derivatives. The combined chemical and biotechnological methods can be applied to obtain such compounds. In the present study, 2′-hydroxy-2-methylchalcone was synthesized and biotransformed in the cultures of entomopathogenic filamentous fungi Beauveria bassiana KCH J1.5, Isaria fumosorosea KCH J2 and Isaria farinosa KCH J2.6, which have been known for their extensive enzymatic system and ability to perform glycosylation of flavonoids. As a result, five new glycosylated dihydrochalcones were obtained. Biotransformation of 2′-hydroxy-2-methylchalcone by B. bassiana KCH J1.5 resulted in four glycosylated dihydrochalcones: 2′-hydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside, 2′,3-dihydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside, 2′-hydroxy-2-hydroxymethyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside, and 2′,4-dihydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside. In the culture of I. fumosorosea KCH J2 only one product was formed—3-hydroxy-2-methyldihydrochalcone 2′-O-β-d-(4″-O-methyl)-glucopyranoside. Biotransformation performed by I. farinosa KCH J2.6 resulted in the formation of two products: 2′-hydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside and 2′,3-dihydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside. The structures of all obtained products were established based on the NMR spectroscopy. All products mentioned above may be used in further studies as potentially bioactive compounds with improved stability and bioavailability. These compounds can be considered as flavor enhancers and potential sweeteners.     

Glycosylation of Methylflavonoids in the Cultures of Entomopathogenic Filamentous Fungi as a Tool for Obtaining New Biologically Active Compounds

Flavonoid compounds are secondary plant metabolites with numerous biological activities; they naturally occur mainly in the form of glycosides. The glucosyl moiety attached to the flavonoid core makes them more stable and water-soluble. The methyl derivatives of flavonoids also show increased stability and intestinal absorption. Our study showed that such flavonoids can be obtained by combined chemical and biotechnological methods with entomopathogenic filamentous fungi as glycosylation biocatalysts. In the current paper, two flavonoids, i.e., 2′-hydroxy-4-methylchalcone and 4′-methylflavone, have been synthesized and biotransformed in the cultures of two strains of entomopathogenic filamentous fungi Isaria fumosorosea KCH J2 and Beauveria bassiana KCH J1.5. Biotransformation of 2′-hydroxy-4-methylchalcone resulted in the formation of two dihydrochalcone glucopyranoside derivatives in the culture of I. fumosorosea KCH J2 and chalcone glucopyranoside derivative in the case of B. bassiana KCH J1.5. 4′-Methylflavone was transformed in the culture of I. fumosorosea KCH J2 into four products, i.e., 4′-hydroxymethylflavone, flavone 4′-methylene-O-β-d-(4″-O-methyl)-glucopyranoside, flavone 4′-carboxylic acid, and 4′-methylflavone 3-O-β-d-(4″-O-methyl)-glucopyranoside. 4′-Methylflavone was not efficiently biotransformed in the culture of B. bassiana KCH J1.5. The computer-aided simulations based on the chemical structures of the obtained compounds showed their improved physicochemical properties and antimicrobial, anticarcinogenic, hepatoprotective, and cardioprotective potential.     

4′-Methylflavanone Glycosides Obtained Using Biotransformation in the Entomopathogenic Filamentous Fungi Cultures as Potential Anticarcinogenic,Antimicrobial, and Hepatoprotective Agents

Flavonoid compounds exhibit numerous biological activities and significantly impact human health. The presence of methyl or glucosyl moieties attached to the flavonoid core remarkably modifies their physicochemical properties and improves intestinal absorption. Combined chemical and biotechnological methods can be applied to obtain such derivatives. In the presented study, 4′-methylflavanone was synthesized and biotransformed in the cultures of three strains of entomopathogenic filamentous fungi, i.e., Isaria fumosorosea KCH J2, Beauveria bassiana KCH J1.5, and Isaria farinosa KCH J2.1. The microbial transformation products in the culture of I. fumosorosea KCH J2, flavanone 4′-methylene-O-β-D-(4″-O-methyl)-glucopyranoside, 2-phenyl-(4′-hydroxymethyl)-4-hydroxychromane, and flavanone 4′-carboxylic acid were obtained. Biotransformation of 4′-methylflavanone in the culture of B. bassiana KCH J1.5 resulted in the formation of one main product, i.e., flavanone 4′-methylene-O-β-D-(4″-O-methyl)-glucopyranoside. In the case of I. farinosa KCH J2.6 as a biocatalyst, three products, i.e., flavanone 4′-methylene-O-β-D-(4″-O-methyl)-glucopyranoside, flavanone 4′-carboxylic acid, and 4′-hydroxymethylflavanone 4-O-β-D-(4″-O-methyl)-glucopyranoside were obtained. The Swiss-ADME online simulations confirmed the increase in water solubility of 4′-methylflavanone glycosides and analyses performed using the Way2Drug Pass Online prediction tool indicated that flavanone 4′-methylene-O-β-D-(4″-O-methyl)-glucopyranoside and 4′-hydroxymethylflavanone 4-O-β-D-(4″-O-methyl)-glucopyranoside, which had not been previously reported in the literature, are promising anticarcinogenic, antimicrobial, and hepatoprotective agents.     

The Effect of the Aerial Part of Lindera akoensis on Lipopolysaccharides (LPS)-Induced Nitric Oxide Production in RAW264.7 Cells

Four new secondary metabolites, 3α-((E)-Dodec-1-enyl)-4β-hydroxy-5β-methyldihydrofuran-2-one (1), linderinol (6), 4′-O-methylkaempferol 3-O-α-l-(4″-E-p-coumaroyl)rhamnoside (11) and kaempferol 3-O-α-l-(4″-Z-p-coumaroyl) rhamnoside (12) with eleven known compounds—3-epilistenolide D₁ (2), 3-epilistenolide D₂ (3), (3Z,4α,5β)-3-(dodec-11-ynylidene)-4-hydroxy-5-methylbutanolide (4), (3E,4β,5β)-3-(dodec-11-ynylidene)-4-hydroxy-5-methylbutanolide (5), matairesinol (7), syringaresinol (8), (+)-pinoresinol (9), salicifoliol (10), 4″-p-coumaroylafzelin (13), catechin (14) and epicatechin (15)—were first isolated from the aerial part of Lindera akoensis. Their structures were determined by detailed analysis of 1D- and 2D-NMR spectroscopic data. All of the compounds isolated from Lindera akoensis showed that in vitro anti-inflammatory activity decreases the LPS-stimulated production of nitric oxide (NO) in RAW 264.7 cell, with IC₅₀ values of 4.1–413.8 μM.     

Chemical Constituents Analysis and Antidiabetic Activity Validation of Four Fern Species from Taiwan

Pterosins are abundant in ferns, and pterosin A was considered a novel activator of adenosine monophosphate-activated protein kinase, which is crucial for regulating blood glucose homeostasis. However, the distribution of pterosins in different species of ferns from various places in Taiwan is currently unclear. To address this question, the distribution of pterosins, glucose-uptake efficiency, and protective effects of pterosin A on β-cells were examined. Our results showed that three novel compounds, 13-chloro-spelosin 3-O-β-d-glucopyranoside (1), (3R)-Pterosin D 3-O-β-d-(3''-p-coumaroyl)-glucopyranoside (2), and (2R,3R)-Pterosin L 3-O-β-d-(3''-p-coumaroyl)-glucopyranoside (3), were isolated for the first time from four fern species (Ceratopteris thalictroides, Hypolepis punctata, Nephrolepis multiflora, and Pteridium revolutum) along with 27 known compounds. We also examined the distribution of these pterosin compounds in the mentioned fern species (except N. multiflora). Although all pterosin analogs exhibited the same effects in glucose uptake assays, pterosin A prevented cell death and reduced reactive oxygen species (ROS) production. This paper is the first report to provide new insights into the distribution of pterosins in ferns from Taiwan. The potential anti-diabetic activity of these novel phytocompounds warrants further functional studies.     

Phenolic Compounds from Halimodendron halodendron (Pall.) Voss and Their Antimicrobial and Antioxidant Activities

Halimodendron halodendron has been used as forage in northwestern China for a long time. Its young leaves and flowers are edible and favored by indigenous people. In this study, eleven phenolic compounds were bioassay-guided and isolated from the aerial parts of H. halodendron for the first time. They were identified by means of physicochemical and spectrometric analysis as quercetin (1), 3,5,7,8,4′-pentahydroxy-3′-methoxy flavone (2), 3-O-methylquercetin (3), 3,3′-di-O-methylquercetin (4), 3,3′-di-O-methylquercetin-7-O-β-d-glucopyranoside (5), isorhamentin-3-O-β-d-rutinoside (6), 8-O-methylretusin (7), 8-O-methylretusin-7-O-β-d-glucopyranoside (8), salicylic acid (9), p-hydroxybenzoic acid (ferulic acid) (10), and 4-hydroxy-3-methoxy cinnamic acid (11). They were sorted as flavonols (1–6), soflavones (7 and 8), and phenolic acids (9–11). Among the compounds, flanools 1–4 revealed a strong antibacterial activity with minimum inhibitory concentration (MIC) values of 50–150 μg/mL, and median inhibitory concentration (IC₅₀) values of 26.8–125.1 μg/mL. The two isoflavones (7 and 8) showed moderate inhibitory activity on the test bacteria. Three phenolic acids (9, 10 and 11) showed strong antibacterial activity with IC₅₀ values of 28.1–149.7 μg/mL. Antifungal activities of the compounds were similar to their antibacterial activities. All these phenolic compounds showed significant antimicrobial activity with a broad spectrum as well as antioxidant activity based on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and β-carotene-linoleic acid bleaching assays. In general, the flavonol aglycones with relatively low polarity exhibited stronger activities than the glycosides. The results suggest the potential of this plant as a source of functional food ingredients and provide support data for its utilization as forage as well.     

Hydrogen Bonding Drives Helical Chirality via 10-Membered Rings in Dipeptide Conjugates of Ferrocene-1,1′-Diamine

Considering the enormous importance of protein turns as participants in various biological events, such as protein–protein interactions, great efforts have been made to develop their conformationally and proteolytically stable mimetics. Ferrocene-1,1′-diamine was previously shown to nucleate the stable turn structures in peptides prepared by conjugation with Ala (III) and Ala–Pro (VI). Here, we prepared the homochiral conjugates of ferrocene-1,1′-diamine with l-/d-Phe (32/35), l-/d-Val (33/36), and l-/d-Leu (34/37) to investigate (1) whether the organometallic template induces the turn structure upon conjugation with amino acids, and (2) whether the bulky or branched side chains of Phe, Val, and Leu affect hydrogen bonding. Detailed spectroscopic (IR, NMR, CD), X-ray, and DFT studies revealed the presence of two simultaneous 10-membered interstrand hydrogen bonds, i.e., two simultaneous β-turns in goal compounds. A preliminary biological evaluation of d-Leu conjugate 37 showed its modest potential to induce cell cycle arrest in the G0/G1 phase in the HeLa cell line but these results need further investigation.     

Structure and Antitumor and Immunomodulatory Activities of a Water-Soluble Polysaccharide from Dimocarpus longan Pulp

A new water-soluble polysaccharide (longan polysaccharide 1 (LP1)) was extracted and successfully purified from Dimocarpus longan pulp via diethylaminoethyl (DEAE)-cellulose anion-exchange and Sephacryl S-300 HR gel chromatography. The chemical structure was determined using Infrared (IR), gas chromatography (GC) and nuclear magnetic resonance (NMR) analysis. The results indicated that the molecular weight of the sample was 1.1 × 10⁵ Da. Monosaccharide composition analysis revealed that LP1 was composed of Glc, GalA, Ara and Gal in a molar ratio of 5.39:1.04:0.74:0.21. Structural analysis indicated that LP1 consisted of a backbone of →4)-α-d-Glcp-(1→4)-α-d-GalpA-(1→4)-α-d-Glcp-(1→4)-β-d-Glcp-(1→ units with poly saccharide side chains composed of →2)-β-d-Fruf-(1→2)-l-sorbose-(1→ attached to the O-6 position of the α-d-Glcp residues. In vitro experiments indicated that LP1 had significantly high antitumor activity against SKOV3 and HO8910 tumor cells, with inhibition percentages of 40% and 50%, respectively. In addition, LP1 significantly stimulated the production of the cytokine interferon-γ (IFN-γ), increased the activity of murine macrophages and enhanced B- and T-lymphocyte proliferation. The results of this study demonstrate that LP1 has potential applications as a natural antitumor agent with immunomodulatory activity.     

New Triterpenoids with Cytotoxic Activity from Actinidia Valvata

Two new triterpenoids, 30-O-β-d-glucopyranosyloxy-2α,3α,24-trihydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester (1) and 2α,3β,3,30-tetrahydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester (2) were isolated from roots of Actinidia valvata Dunn. Their structures were elucidated by means of extensive spectroscopic studies. Both these two new compounds showed moderate cytotoxic activity in vitro against BEL-7402 and SMMC-7721 tumor cell line.     

Trehalose Analogues: Latest Insights in Properties and Biocatalytic Production

Trehalose (α-d-glucopyranosyl α-d-glucopyranoside) is a non-reducing sugar with unique stabilizing properties due to its symmetrical, low energy structure consisting of two 1,1-anomerically bound glucose moieties. Many applications of this beneficial sugar have been reported in the novel food (nutricals), medical, pharmaceutical and cosmetic industries. Trehalose analogues, like lactotrehalose (α-d-glucopyranosyl α-d-galactopyranoside) or galactotrehalose (α-d-galactopyranosyl α-d-galactopyranoside), offer similar benefits as trehalose, but show additional features such as prebiotic or low-calorie sweetener due to their resistance against hydrolysis during digestion. Unfortunately, large-scale chemical production processes for trehalose analogues are not readily available at the moment due to the lack of efficient synthesis methods. Most of the procedures reported in literature suffer from low yields, elevated costs and are far from environmentally friendly. “Greener” alternatives found in the biocatalysis field, including galactosidases, trehalose phosphorylases and TreT-type trehalose synthases are suggested as primary candidates for trehalose analogue production instead. Significant progress has been made in the last decade to turn these into highly efficient biocatalysts and to broaden the variety of useful donor and acceptor sugars. In this review, we aim to provide an overview of the latest insights and future perspectives in trehalose analogue chemistry, applications and production pathways with emphasis on biocatalysis.     

Structural Determination and Genetic Identification of the O-Antigen from an Escherichia coli Strain,LL004, Representing a Novel Serogroup

The O-antigen is the outermost component of the lipopolysaccharide layer in Gram-negative bacteria, and the variation of O-antigen structure provides the basis for bacterial serological diversity. Here, we determined the O-antigen structure of an Escherichia coli strain, LL004, which is totally different from all of the E. coli serogroups. The tetrasaccharide repeating unit was determined as →4)-β-d-Galp-(1→3)-β-d-GlcpNAc6OAc(~70%)-(1→3)-β-d-GalpA-(1→3)-β-d-GalpNAc-(1→ with monosaccharide analysis and NMR spectra. We also characterized the O-antigen gene cluster of LL004, and sequence analysis showed that it correlated well with the O-antigen structure. Deletion and complementation testing further confirmed its role in O-antigen biosynthesis, and indicated that the O-antigen of LL004 is assembled via the Wzx/Wzy dependent pathway. Our findings, in combination, suggest that LL004 should represent a novel serogroup of E. coli.     

Inhibitory Effect of (2R)-4-(4-hydroxyphenyl)-2-butanol 2-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside on RANKL-Induced Osteoclast Differentiation and ROS Generation in Macrophages

In bone homeostasis, bone loss due to excessive osteoclasts and inflammation or osteolysis in the bone formation process cause bone diseases such as osteoporosis. Suppressing the accompanying oxidative stress such as ROS in this process is an important treatment strategy for bone disease. Therefore, in this study, the effect of (2R)-4-(4-hydroxyphenyl)-2-butanol 2-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (BAG), an arylbutanoid glycoside isolated from Betula platyphylla var. japonica was investigated in RANKL-induced RAW264.7 cells and LPS-stimulated MC3E3-T1 cells. BAG inhibited the activity of TRAP, an important marker of osteoclast differentiation and F-actin ring formation, which has osteospecific structure. In addition, the protein and gene levels were suppressed of integrin β3 and CCL4, which play an important role in the osteoclast-induced bone resorption and migration of osteoclasts, and inhibited the production of ROS and restored the expression of antioxidant enzymes such as SOD and CAT lost by RANKL. The inhibitory effect of BAG on osteoclast differentiation and ROS production appears to be due to the inhibition of MAPKs phosphorylation and NF-κβ translocation, which play a major role in osteoclast differentiation. In addition, BAG inhibited ROS generated by LPS and effectively restores the mineralization of lost osteoblasts, thereby showing the effect of bone formation in the inflammatory situation accompanying bone loss by excessive osteoclasts, suggesting its potential as a new natural product-derived bone disease treatment.     

Identification of Human UDP-Glucuronosyltransferase 1A4 as the Major Isozyme Responsible for the Glucuronidation of 20(S)-Protopanaxadiol in Human Liver Microsomes

20(S)-protopanaxadiol (PPD), one of the representative aglycones of ginsenosides, has a broad spectrum of pharmacological activities. Although phase I metabolism has been investigated extensively, information regarding phase II metabolism of this compound remains to be elucidated. Here, a glucuronidated metabolite of PPD in human liver microsomes (HLMs) and rat liver microsomes (RLMs) was unambiguously identified as PPD-3-O-β-d-glucuronide by nuclear magnetic resonance spectroscopy and high resolution mass spectrometry. The chemical inhibition and recombinant human UDP-Glucuronosyltransferase (UGT) isoforms assay showed that the PPD glucuronidation was mainly catalyzed by UGT1A4 in HLM, whereas UGT1A3 showed weak catalytic activity. In conclusion, PPD-3-O-β-d-glucuronide was first identified as the principal glucuronidation metabolite of PPD in HLMs, which was catalyzed by UGT1A4.     

Identification and Characterization of a Thermostable GH36 α-Galactosidase from Anoxybacillus vitaminiphilus WMF1 and Its Application in Synthesizing Isofloridoside by Reverse Hydrolysis

An α-galactosidase-producing strain named Anoxybacillus vitaminiphilus WMF1, which catalyzed the reverse hydrolysis of d-galactose and glycerol to produce isofloridoside, was isolated from soil. The α-galactosidase (galV) gene was cloned and expressed in Escherichia coli. The galV was classified into the GH36 family with a molecular mass of 80 kDa. The optimum pH and temperature of galV was pH 7.5 and 60 °C, respectively, and it was highly stable at alkaline pH (6.0–9.0) and temperature below 65 °C. The specificity for p-nitrophenyl α-d-galactopyranoside was 70 U/mg, much higher than that for raffinose and stachyose. Among the metals and reagents tested, galV showed tolerance in the presence of various organic solvents. The kinetic parameters of the enzyme towards p-nitrophenyl α-d-galactopyranoside were obtained as K_m (0.12 mM), V_max (1.10 × 10⁻³ mM s⁻¹), and K_cat/K_m (763.92 mM⁻¹ s⁻¹). During the reaction of reverse hydrolysis, the enzyme exhibited high specificity towards the glycosyl donor galactose and acceptors glycerol, ethanol and ethylene glycol. Finally, the isofloridoside was synthesized using galactose as the donor and glycerol as the acceptor with a 26.6% conversion rate of galactose. This study indicated that galV might provide a potential enzyme source in producing isofloridoside because of its high thermal stability and activity.     

Purification and Structural Identification of Polysaccharides from Bamboo Shoots (Dendrocalamus latiflorus)

Three kinds of polysaccharides, namely, BSP1A, BSP2A, and BSP3B, were isolated from raw bamboo shoot (Dendrocalamus latiflorus) after purification and classification by DEAE cellulose-52 (ion-exchange chromatography) and Sephadex G-50. The molecular weights of BSP1A, BSP2A, and BSP3B were 10.2, 17.0 and 20.0 kDa, respectively, which were measured through GPC (gel performance chromtatography) methods. BSP1A contained arabinose, glucose, and galactose in a molar ratio of 1.0:40.6:8.7. BSP2A and BSP3B contained arabinose, xylose, glucose, and galactose in molar ratios of 6.6:1.0:5.2:10.4 and 8.5:1.0:5.1:11.1, respectively. The existence of the O-glycopeptide bond in BSP1A, BSP2A, and BSP3B was demonstrated by β-elimination reaction. FTIR spectra of the three polysaccharides showed that both BSP2A and BSP3B contained β-d-pyranose sugar rings. However, BSP1A exhibited both β-d-pyranose and α-d-pyranose sugar rings. Congo red test indicated that BSP1A and BSP2A displayed triple helix structures, but BSP3B did not. NMR spectroscopy revealed that BSP1A may exhibit a β-1,6-Glucan pyran type as the main link, and few 1,6-glycosidic galactose pyranose and arabinose bonds were connected; BSP2A mainly demonstrated →5)β-Ara(1→and→3)β-Gal(1→connection. Furthermore, BSP3B mainly presented →3)β-Glu(1→and→3)β-Gal(1→connection and may also contain few other glycosidic bonds.     

2-Acetamido-2-deoxy-d-glucono-1,5-lactone Sulfonylhydrazones: Synthesis and Evaluation as Inhibitors of Human OGA and HexB Enzymes

Inhibition of the human O-linked β-N-acetylglucosaminidase (hOGA, GH84) enzyme is pharmacologically relevant in several diseases such as neurodegenerative and cardiovascular disorders, type 2 diabetes, and cancer. Human lysosomal hexosaminidases (hHexA and hHexB, GH20) are mechanistically related enzymes; therefore, selective inhibition of these enzymes is crucial in terms of potential applications. In order to extend the structure–activity relationships of OGA inhibitors, a series of 2-acetamido-2-deoxy-d-glucono-1,5-lactone sulfonylhydrazones was prepared from d-glucosamine. The synthetic sequence involved condensation of N-acetyl-3,4,6-tri-O-acetyl-d-glucosamine with arenesulfonylhydrazines, followed by MnO₂ oxidation to the corresponding glucono-1,5-lactone sulfonylhydrazones. Removal of the O-acetyl protecting groups by NH₃/MeOH furnished the test compounds. Evaluation of these compounds by enzyme kinetic methods against hOGA and hHexB revealed potent nanomolar competitive inhibition of both enzymes, with no significant selectivity towards either. The most efficient inhibitor of hOGA was 2-acetamido-2-deoxy-d-glucono-1,5-lactone 1-naphthalenesulfonylhydrazone (5f, K_i = 27 nM). This compound had a K_i of 6.8 nM towards hHexB. To assess the binding mode of these inhibitors to hOGA, computational studies (Prime protein–ligand refinement and QM/MM optimizations) were performed, which suggested the binding preference of the glucono-1,5-lactone sulfonylhydrazones in an s-cis conformation for all test compounds.     

Structural Characterization of a Water-Soluble Polysaccharide from the Fruiting Bodies of Agaricus bisporus

An edible fungal polysaccharide termed as ABP was obtained by extraction with hot water, and followed successive chromatographic purification using DEAE-Sepharose Fast Flow column and Sephacryl S-300 High-Resolution column. A symmetrical peak was obtained on high-performance size-exclusion chromatography with an average molecular weight of 5.17 × 10⁴ Da, which was named ABP, and its main components were d-glucose and d-mannose. Based on the study of methylation analysis, along with FT-IR, GC, GC-MS, 1D ¹H and ¹³C NMR and 2D NMR (H-HCOSY, TOCSY, HMQC, and NOESY), its chemical structure was featured with a repeating unit (1→6) linking β-d-Glcp as the main backbone with (1→4)-linked α-d-Manp units. The structure of the mainly repeating units of ABP was established as:  → 6) - β - D - Glucp - (1 → 4) - α - D - Manp(1 → 6) - β - D - Glucp - (1 → 6) - β - D - Glucp - (1 →      

Structural Basis of Cysteine Ligase MshC Inhibition by Cysteinyl-Sulfonamides

Mycothiol (MSH), the major cellular thiol in Mycobacterium tuberculosis (Mtb), plays an essential role in the resistance of Mtb to various antibiotics and oxidative stresses. MshC catalyzes the ATP-dependent ligation of 1-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-d-myo-inositol (GlcN-Ins) with l-cysteine (l-Cys) to form l-Cys-GlcN-Ins, the penultimate step in MSH biosynthesis. The inhibition of MshC is lethal to Mtb. In the present study, five new cysteinyl-sulfonamides were synthesized, and their binding affinity with MshC was evaluated using a thermal shift assay. Two of them bind the target with EC₅₀ values of 219 and 231 µM. Crystal structures of full-length MshC in complex with these two compounds showed that they were bound in the catalytic site of MshC, inducing dramatic conformational changes of the catalytic site compared to the apo form. In particular, the observed closure of the KMSKS loop was not detected in the published cysteinyl-sulfamoyl adenosine-bound structure, the latter likely due to trypsin treatment. Despite the confirmed binding to MshC, the compounds did not suppress Mtb culture growth, which might be explained by the lack of adequate cellular uptake. Taken together, these novel cysteinyl-sulfonamide MshC inhibitors and newly reported full-length apo and ligand-bound MshC structures provide a promising starting point for the further development of novel anti-tubercular drugs targeting MshC.     

Heterogenicity within the LPS Structure in Relation to the Chosen Genomic and Physiological Features of the Plant Pathogen Pectobacterium parmentieri

Pectobacterium parmentieri is a pectinolytic plant pathogenic bacterium causing high economic losses of cultivated plants. The highly devastating potential of this phytopathogen results from the efficient production of plant cell wall-degrading enzymes, i.e., pectinases, cellulases and proteases, in addition to the impact of accessory virulence factors such as motility, siderophores, biofilm and lipopolysaccharide (LPS). LPS belongs to pathogen-associated molecular patterns (PAMPs) and plays an important role in plant colonization and interaction with the defense systems of the host. Therefore, we decided to investigate the heterogeneity of O-polysaccharides (OPS) of LPS of different strains of P. parmentieri, in search of an association between the selected genomic and phenotypic features of the strains that share an identical structure of the OPS molecule. In the current study, OPS were isolated from the LPS of two P. parmentieri strains obtained either in Finland in the 1980s (SCC3193) or in Poland in 2013 (IFB5432). The purified polysaccharides were analyzed by utilizing 1D and 2D NMR spectroscopy (¹H, DQF-COSY, TOCSY, ROESY, HSQC, HSQC-TOCSY and HMBC) in addition to chemical methods. Sugar and methylation analyses of native polysaccharides, absolute configuration assignment of constituent monosaccharides and NMR spectroscopy data revealed that these two P. parmentieri strains isolated in different countries possess the same structure of OPS with a very rare residue of 5,7-diamino-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid (pseudaminic acid) substituted in the position C-8: →3)-β-d-Galf-(1→3)-α-d-Galp-(1→8)-β-Pse4Ac5Ac7Ac-(2→6)-α-d-Glcp-(1→6)-β-d-Glcp-(1→. The previous study indicated that three other P. parmentieri strains, namely IFB5427, IFB5408 and IFB5443, exhibit a different OPS molecule than SCC3193 and IFB5432. The conducted biodiversity-oriented assays revealed that the P. parmentieri IFB5427 and IFB5408 strains possessing the same OPS structure yielded the highest genome-wide similarity, according to average nucleotide identity analyses, in addition to the greatest ability to macerate chicory tissue among the studied P. parmentieri strains. The current research demonstrated a novel OPS structure, characteristic of at least two P. parmentieri strains (SCC3193 and IFB5432), and discussed the observed heterogenicity in the OPS of P. parmentieri in a broad genomic and phenotype-related context.     

Tryptophanase-Catalyzed l-Tryptophan Synthesis from d-Serine in the Presence of Diammonium Hydrogen Phosphate

Tryptophanase, an enzyme with extreme absolute stereospecificity for optically active stereoisomers, catalyzes the synthesis of l-tryptophan from l-serine and indole through a β-substitution mechanism of the ping-pong type, and has no activity on d-serine. We previously reported that tryptophanase changed its stereospecificity to degrade d-tryptophan in highly concentrated diammonium hydrogen phosphate, (NH₄)₂HPO₄ solution. The present study provided the same stereospecific change seen in the d-tryptophan degradation reaction also occurs in tryptophan synthesis from d-serine. Tryptophanase became active to d-serine to synthesize l-tryptophan in the presence of diammonium hydrogen phosphate. This reaction has never been reported before. d-serine seems to undergo β-replacement via an enzyme-bonded α-aminoacylate intermediate to yield l-tryptophan.